Yoshihiro Kobae

Deletion of a Histidine-rich Loop of AtMTP1, a Vacuolar Zn2+/H+ Antiporter of Arabidopsis thaliana, Stimulates the Transport Activity

Arabidopsis thaliana AtMTP1 belongs to the cation diffusion facilitator family and is localized on the vacuolar membrane. We investigated the enzymatic kinetics of AtMTP1 by a heterologous expression system in the yeast Saccharomyces cerevisiae, which lacked genes for vacuolar membrane zinc transporters ZRC1 and COT1. The yeast mutant expressing AtMTP1 heterologously was tolerant to 10 mm ZnCl2. Active transport of zinc into vacuoles of living yeast cells expressing AtMTP1 was confirmed by the fluorescent zinc indicator FuraZin-1. Zinc transport was quantitatively analyzed by using vacuolar membrane vesicles prepared from AtMTP1-expressing yeast cells and radioisotope 65Zn2+. Active zinc uptake depended on a pH gradient generated by endogenous vacuolar H+-ATPase. The activity was inhibited by bafilomycin A1, an inhibitor of the H+-ATPase. The Km for Zn2+ and Vmax of AtMTP1 were determined to be 0.30 μm and 1.22 nmol/min/mg, respectively. We prepared a mutant AtMTP1 that lacked the major part (32 residues from 185 to 216) of a long histidine-rich hydrophilic loop in the central part of AtMTP1. Yeast cells expressing the mutant became hyperresistant to high concentrations of Zn2+ and resistant to Co2+. The Km and Vmax values were increased 2–11-fold. These results indicate that AtMTP1 functions as a Zn2+/H+ antiporter in vacuoles and that a histidine-rich region is not essential for zinc transport. We propose that a histidine-rich loop functions as a buffering pocket of Zn2+ and a sensor of the zinc level at the cytoplasmic surface. This loop may be involved in the maintenance of the level of cytoplasmic Zn2+.

Dynamics of Periarbuscular Membranes Visualized with a Fluorescent Phosphate Transporter in Arbuscular Mycorrhizal Roots of Rice

In arbuscular mycorrhizal (AM) symbiosis, host plants supply photosynthates to AM fungi and, in return, they receive inorganic nutrients such as phosphate from finely branched fungal arbuscules. Plant cortical cells envelope arbuscules with periarbuscular membranes that are continuous with the plant plasma membranes. We prepared transgenic rice (Oryza sativa) plants that express a fusion of green fluorescent protein with rice AM-inducible phosphate transporter, OsPT11-GFP, and grew them with AM fungi. The fluorescence of the fusion transporter was observed in the arbuscule branch domain, where active nutrient exchange seems to occur. In contrast, a signal was not detected around intracellular hyphal coils on colonization by either Glomus mosseae or Gigaspora rosea, making the difference between Arum- and Paris-type mycorrhizae ambiguous. We also invented a simple device involving glass-bottomed Petri dishes for in planta observation of fluorescent proteins in living AM roots with an inverted fluorescence microscope. The plant bodies remain completely intact, avoiding any stressful procedure such as cutting, staining, etc. Since rice roots exhibit a very low level of autofluorescence, the device enabled clear time-lapse imaging to analyze the formation, function and degeneration of arbuscules. In cortical cells, arbuscules seemed to be functional for only 2-3 d. Suddenly, the arbuscular branches became fragile and they shrank. At this stage, however, the periarbuscular membranes appeared intact. Then, the fluorescence of the transporter disappeared within only 2.5-5.5 h. The collapse of arbuscules occurred in the subsequent several days. Thus, our device has a great advantage for investigation of dynamic features of AM symbiosis.

Localized Expression of Arbuscular Mycorrhiza-Inducible Ammonium Transporters in Soybean

The majority of land plants acquire soil nutrients, such as phosphorus and nitrogen, not only through the root surface but also through arbuscular mycorrhizal (AM) fungi. Soybean is the most important leguminous crop in the world. We found 16 ammonium transporter genes in the soybean genome, five of which are AM inducible. Among them, promoter-reporter analysis indicated that the most abundantly transcribed gene, GmAMT4.1, showed specific expression in arbusculated cortical cells. Moreover, the GmAMT4.1-green fluorescent protein fusion was localized on the branch domain of periarbuscular membranes but not on the trunk region, indicating that active ammonium transfer occurs around the arbuscule branches.

The Bifunctional Plant Receptor, OsCERK1, Regulates Both Chitin-Triggered Immunity and Arbuscular Mycorrhizal Symbiosis in Rice

Plants are constantly exposed to threats from pathogenic microbes and thus developed an innate immune system to protect themselves. On the other hand, many plants also have the ability to establish endosymbiosis with beneficial microbes such as arbuscular mycorrhizal (AM) fungi or rhizobial bacteria, which improves the growth of host plants. How plants evolved these systems managing such opposite plant-microbe interactions is unclear. We show here that knockout (KO) mutants of OsCERK1, a rice receptor kinase essential for chitin signaling, were impaired not only for chitin-triggered defense responses but also for AM symbiosis, indicating the bifunctionality of OsCERK1 in defense and symbiosis. On the other hand, a KO mutant of OsCEBiP, which forms a receptor complex with OsCERK1 and is essential for chitin-triggered immunity, established mycorrhizal symbiosis normally. Therefore, OsCERK1 but not chitin-triggered immunity is required for AM symbiosis. Furthermore, experiments with chimeric receptors showed that the kinase domains of OsCERK1 and homologs from non-leguminous, mycorrhizal plants could trigger nodulation signaling in legume-rhizobium interactions as the kinase domain of Nod factor receptor1 (NFR1), which is essential for triggering the nodulation program in leguminous plants, did. Because leguminous plants are believed to have developed the rhizobial symbiosis on the basis of AM symbiosis, our results suggest that the symbiotic function of ancestral CERK1 in AM symbiosis enabled the molecular evolution to leguminous NFR1 and resulted in the establishment of legume-rhizobia symbiosis. These results also suggest that OsCERK1 and homologs serve as a molecular switch that activates defense or symbiotic responses depending on the infecting microbes.

A Mutant Strain Arabidopsis thaliana that Lacks Vacuolar Membrane Zinc Transporter MTP1 Revealed the Latent Tolerance to Excessive Zinc

A mutant line of Arabidopsis thaliana that lacks a vacuolar membrane Zn(2+)/H(+) antiporter MTP1 is sensitive to zinc. We examined the physiological changes in this loss-of-function mutant under high-Zn conditions to gain an understanding of the mechanism of adaptation to Zn stress. When grown in excessive Zn and observed using energy-dispersive X-ray analysis, wild-type roots were found to accumulate Zn in vacuolar-like organelles but mutant roots did not. The Zn content of mutant roots, determined by chemical analysis, was one-third that of wild-type roots grown in high-Zn medium. Severe inhibition of root growth was observed in mtp1-1 seedlings in 500 muM ZnSO(4). Suppression of cell division and elongation by excessive Zn was reversible and the cells resumed growth in normal medium. In mutant roots, a marked formation of reactive oxygen species (ROS) appeared in the meristematic zone, where the MTP1 gene was highly expressed. Zn treatment enhanced the expression of several genes involved in Zn tolerance: namely, the plasma membrane Zn(2+)-export ATPase, HMA4, and plasma and vacuolar membrane proton pumps. CuZn-superoxide dismutases, involved in the detoxification of ROS, were also induced. The expression of plasma membrane Zn-uptake transporter, ZIP1, was suppressed. The up- or down-regulation of these genes might confer the resistance to Zn toxicity. These results indicate an essential role of MTP1 in detoxification of excessive Zn and provide novel information on the latent adaptation mechanism to Zn stress, which is hidden by MTP1.

Interactions between plants and arbuscular mycorrhizal fungi.

Arbuscular mycorrhizal (AM) fungi inhabit the root cortical cells of most plants and obtain photosynthates from the host plants while they transfer mineral nutrients from the soil to the hosts. In this review, we first summarize recent progress regarding signal molecules involved in the recognition of each symbiont, the signaling pathways in the host plants, and the characteristics of AM-inducible nutrient transporters, which were elucidated mainly using model legumes. Then, we summarize studies on the colonization by AM fungi of lower plants and of the roots of major crops. There are not only AM-responsive crops like maize, sorghum, and soybean but also AM-nonresponsive ones like wheat, barley, and rice. Finally, we mention the worldwide problems of limited and biased agricultural resources and discuss future directions as to how we can make use of AM symbiosis for improving crop production and establishing sustainable agriculture.

Identification and Expression Analysis of Arbuscular Mycorrhiza-Inducible Phosphate Transporter Genes of Soybean

Soybeans, the world’s leading leguminous crop, establish mutualistic symbiosis with arbuscular mycorrhizal (AM) fungi. AM fungi colonize root cortical cells forming arbuscules, highly branched fungal structures. Arbuscules are enveloped by plant-derived periarbuscular membranes through which plants obtain mineral nutrients, particularly phosphate. We searched the soybean genome in silico, and found 14 Pht1 genes encoding phosphate transporters putatively localized on the plasma membranes. Time course analyses involving reverse transcription-PCR indicated that three of these were AM-inducible. GmPT10 and GmPT11 were induced on fungal colonization, while a transcript of GmPT7 appeared in the later stages. The transport activities of GmPT10 and GmPT11 were confirmed by complementation of a yeast mutant. Soybean hairy roots expressing the GmPT10-green fluorescent protein (GFP) or GmPT11-GFP fusion protein under the control of corresponding promoter showed GFP fluorescence on the branch domains of periarbuscular membranes, indicating that active phosphate transport occurred there.

Amino acid screening based on structural modeling identifies critical residues for the function, ion selectivity and structure of Arabidopsis MTP1.

Arabidopsisu2003thaliana MTP1 is a vacuolar membrane Zn2+/H+ antiporter of the cation diffusion facilitator family. Here we present a structure–function analysis of AtMTP1‐mediated transport and its remarkable Zn2+ selectivity by functional complementation tests of more than 50 mutant variants in metal‐sensitive yeast strains. This was combined with homology modeling of AtMTP1 based on the crystal structure of the Escherichiau2003coli broad‐specificity divalent cation transporter YiiP. The Zn2+‐binding sites of EcYiiP in the cytoplasmic C‐terminus, and the pore formed by transmembrane helices TM2 and TM5, are conserved in AtMTP1. Although absent in EcYiiP, Cys31 and Cys36 in the extended N‐terminal cytosolic domain of AtMTP1 are necessary for complementation of a Zn‐sensitive yeast strain. On the cytosolic side of the active Zn2+‐binding site inside the transmembrane pore, Ala substitution of either Asn258 in TM5 or Ser101 in TM2 non‐selectively enhanced the metal tolerance conferred by AtMTP1. Modeling predicts that these residues obstruct the movement of cytosolic Zn2+ into the intra‐membrane Zn2+‐binding site of AtMTP1. A conformational change in the immediately preceding His‐rich cytosolic loop may displace Asn258 and permit Zn2+ entry into the pore. This would allow dynamic coupling of Zn2+ transport to the His‐rich loop, thus acting as selectivity filter or sensor of cytoplasmic Zn2+ levels. Individual mutations at diverse sites within AtMTP1 conferred Co and Cd tolerance in yeast, and included deletions in N‐terminal and His‐rich intra‐molecular cytosolic domains, and mutations of single residues flanking the transmembrane pore or participating in intra‐ or inter‐molecular domain interactions, all of which are not conserved in the non‐selective EcYiiP.

Immunochemical Analysis of Aquaporin Isoforms in Arabidopsis Suspension-Cultured Cells

Aquaporins mediate the movement of water across biomembranes. Arabidopsis thaliana contains 35 aquaporins that belong to four subfamilies (PIP, TIP, SIP, and NIP). We investigated their expression profiles immunochemically in suspension-cultured Arabidopsis thaliana cells during growth and in response to salt and osmotic stresses. Protein amounts of all aquaporins were much lower in cultured cells than in the plant tissues. This is consistent with the low water permeability of protoplasts from cultured cells. After treatment with NaCl, the protein amounts of PIP2;1, PIP2;2, and PIP2;3 in the cells increased several-fold, and those of TIP1;1 and TIP1;2, 15- and 3-fold respectively. PIP1 did not change under the stress. Cell death began after 19 d in culture, accompanied by marked accumulation of PIPs and TIPs and a gradual decrease in SIPs. Our results suggest the followings: (i) Accumulation of aquaporin isoforms was individually regulated at low levels in single cells. (ii) At least PIP2;2, PIP2;3, TIP1;1, and TIP1;2 are stress-responsive aquaporins in suspension cells. (iii) A sudden increment of several members of PIP2 and TIP1 subfamilies might be related to cell death.

Lipid Droplets of Arbuscular Mycorrhizal Fungi Emerge in Concert with Arbuscule Collapse

Plants share photosynthetically fixed carbon with arbuscular mycorrhizal (AM) fungi to maintain their growth and nutrition. AM fungi are oleogenic fungi that contain numerous lipid droplets in their syncytial mycelia during most of their life cycle. These lipid droplets are probably used for supporting growth of extraradical mycelia and propagation; however, when and where the lipid droplets are produced remains unclear. To address these issues, we investigated the correlation between intracellular colonization stages and the appearance of fungal lipid droplets in roots by a combination of vital staining of fungal structures, selective staining of lipids and live imaging. We discovered that a surge of lipid droplets coincided with the collapse of arbuscular branches, indicating that arbuscule collapse and the emergence of lipid droplets may be associated processes. This phenomenon was observed in the model AM fungus Rhizophagus irregularis and the ancestral member of AM fungi Paraglomus occultum. Because the collapsing arbuscules were metabolically inactive, the emerged lipid droplets are probably derived from preformed lipids but not de novo synthesized. Our observations highlight a novel mode of lipid release by AM fungi.